ABSTRACT During the past two decades, prostate cancer (PCa) has become the most commonly diagnosed cancer and ranks as the second leading cause of cancer deaths for American men. Prostate biopsy is the standard procedure for evaluating the presence and aggressiveness of PCa. Guided by transrectal ultrasound (TRUS) and preprocedure magnetic resonance imaging (MRI), small pieces of tissues are removed from the prostate. The architectural heterogeneities are assigned a Gleason score as a quantitative description of the aggressiveness. However, TRUS has low sensitivity to PCa. MRI has limited availability, and a spatial mismatch can occur when co-registering the preprocedure MRI with the real-time TRUS. Therefore, standard TRUS-guided biopsy, either with or without additional MRI, suffers from missing or under-sampling clinically significant tumors, leading to under-grading of PCa. At-risk patients with rising blood-based biomarkers undergo repeated and saturated biopsies, causing extra diagnostic costs and time as well as anxiety and pain. We propose to solve this long-standing technical gap in PCa diagnosis by introducing a needle photoacoustic (PA) probe for online guidance of TRUS biopsy. As validated in our preliminary studies on both animal models and human tissues, quantitative multispectral PA signal analysis possesses the unique capability of objectively characterizing the architectural heterogeneities in prostate tissues and grading PCa in vivo. Our needle PA probe has an all-optical design that allows a small probe dimension to avoid causing additional invasiveness to the current biopsy procedure. Taking advantage of the optical penetration, the needle PA probe can assess the histopathological information in a tissue volume much larger than that of a standard biopsy core without tissue removal. Hence, PA pre-biopsy measurements, performed together with the TRUS needle biopsy, can provide highly valuable diagnostic information covering the whole prostate. Core extractions can then be focused within the suspicious cancerous region(s). The central hypothesis of this research is that a fine needle probe-based PA prostate pre-biopsy can guide prostate biopsy, improve the core yield, and decrease false negative rates. The objective of this study is to validate the correlation between the PA measurements and the PCa grading through an observational human subjects study. The specific aims include investigating the performance of the needle PA probe in accessing PCa using 1) biopsy tissue cores; 2) ex vivo human prostate samples procured through prostatectomy, and 3) human subjects. We will leverage the research team's extensive expertise in the clinical practice of PCa diagnosis and pathology as well as PA technology. The proposed pre-biopsy procedure is designed within the framework of current clinical practice and is, therefore, highly translational. The knowledge gained in this study will prepare us to conduct a future clinical trial of the proposed diagnostic procedure in detecting PCa. Once successfully tested, the PA pre-biopsy will benefit PCa patients by facilitating accurately targeted needle biopsies for the early detection of clinically significant PCa.